Menopause is defined as the permanent cessation of menstruation caused by failure of ovarian follicular development in the presence of elevated gonadotrophin levels. Comprehensive Gynecology (eds. Mishell et al 1997). A hallmark of menopause is the decrease in ovarian follicular estrogen synthesis, although this is only one aspect of the array of physiological events that accompanies the climacteric. Symptoms accompanying menopause vary from woman to woman, but usually include some component of vasomotor instability or hot flashes, often accompanied by psychological symptoms like mood swings and irritability. The loss of estrogen predisposes to coronary artery disease. Loss of estrogen further results in loss of overall bone mineral content caused by an increased resorption of bone without a correlative increase in bone formation. Unabated net loss of bone structural integrity following menopause can culminate in clinically significant osteoporosis. The severity of these climacteric symptoms can be substantially reduced with estrogen replacement therapy. These symptoms represent formidable challenges to the health care system. Recognizing that the current average life expectancy for a woman in the U.S. is 78 years, one can readily calculate that a substantial portion of a woman""s lifespan will be post-menopausal. In 1990, for example, there were more than 50 million women in the U.S. over 50 years of age.
The mean age of physiological menopause in the U.S. is between 51 and 52 years of age, with a range between 45 and 55 years old distributed along a normal curve. Stanford et al., J. Chron. Dis 40:995, 1987. About 10% of women in the U.S. undergo menopause before age 46; 1% of U.S. women enter menopause before age 40. If a woman becomes menopausal before age 40, the condition is termed premature ovarian failure. A variety of factors have been identified that correlate weakly with age of menopause, including number of pregnancies, use of oral contraceptives, duration of lactation, age at menarche, age at last pregnancy, race, height, weight, education or occupational history. Cassou et al., Maturitas 26:165-74, 1997. Cigarette smoking, however, has been observed to decrease the age of onset of menopause by about two years. Menopause may also be therapeutically induced, either chemically, surgically or via radiation (e.g. to reduce the risk of developing breast cancer associated with exposure to estrogens).
Diagnosis of menopause may be made clinically by observing the absence of menses for a year. Most women experience progressive menstrual irregularity that presages menopause. The time between the onset of menstrual irregularity and menopause is called the perimenopause. The median age at onset of perimenopause is 47.5 years; its median length is 4 years. McKinlay et al., Maturitas 14:103, 1992. Prior to the onset of the perimenopause, the length of the menstrual cycle tends to decrease in length, due to the decreased duration of follicular functioning. Munster et al., Br. J. Obstet./Gynaecol. 99:422, 1992. About 10% of women do not enter a perimenopausal phase, rather continuing to have regular cycles until menses suddenly stop.
Hormonal changes often precede changes in menstrual patterns, and early diagnosis for menopause and perimenopause has traditionally involved the measurement of follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels. A significant increase in both FSH and LH levels occurs about 5 years before menopause, with the FSH increase more prominent than the LH increase. Levels of these gonadotrophins peak about one year postmenopausally for LH and two to three years after menopause for FSH. The incidence of ovulatory cycles, measured by elevated luteal phase progesterone levels, decreases from 60% during the 5-6 years before menopause to 5% in the 6 months before menopause. Estrogen levels fall as the number of ovulatory cycles decreases, particularly during the 6-12 months before menopause. FSH release, mainly controlled by inhibin, remains elevated even in the presence of post-menopausal hormone replacement. An initial fall in inhibin level is an early indication of diminishing ovarian function. FSH level rises accordingly, suggesting the woman""s entry into the perimenopausal stage of reproductive function. Initial changes in FSH and inhibin are often transitory. It is therefore important to demonstrate a sustained increase in FSH. This value, combined with low estradiol levels, is used to diagnose the onset of permanent ovarian failure.
Diagnosis of menopause and perimenopause thus tends to be possible only after major changes in the endocrine system have already occurred. A younger woman without premonitory perimenopausal symptoms may have no reason to have her hormone levels measured; she may thus enter early onset menopause (EOM) without any warning. In such a patient, hormonal analysis may not yield a diagnosis until after EOM has taken place. Hormone assays in a younger patient may only be transiently abnormal prior to the onset of permanent ovarian failure. The younger patient destined for EOM is a particularly appropriate subject for early medical intervention (e.g. estrogen replacement therapy). Family planning needs to be considered if early loss of reproductive function is anticipated. Decisions about childbearing and preservation of fertilized ova should be undertaken at an early age in such a patient. Further medical advice is important about the value of hormone replacement therapy for a young woman who faces prolonged post-menopausal levels of estrogen. The relative contribution of estrogen replacement to bone strength and cardiovascular health must be balanced against the possibility of breast cancer development, in response to hormone replacement therapy (HRT).
A complex set of endocrine mechanisms regulates the female reproductive system. Understanding the interrelationship of these mechanisms provides the basis for discerning the factors involved in physiological menopause and EOM. No single organ secretes all the hormones responsible for these processes. The hypothalamus, the pituitary gland, and the ovaries, are primary organs, although adrenal and thyroid hormones also play roles. Feedback mechanisms enable the various hormones to affect the production of other hormones within the reproductive system.
The central nervous system controls reproductive hormone production through its release of gonadotropin-releasing hormone (GnRH). This hormone, produced by the hypothalamus, in turn affects gonadotropin secretion by the pituitary. GnRH secretion is responsive to levels of brain neurotransmitters, in particular the two catecholamines dopamine and norepinephrine. Opioids and prostaglandins in the hypothalamus have also been identified as regulators of GnRH release. The neurotransmitter serotonin has not been associated with GnRH release, but it does stimulate the release of prolactin by the hypothalamus. Other peptides have been identified in the brain that act as neurotransmitters. For example, the peptides activin and inhibin, members of TGF-beta superfamily, have been identified within the brain. These substances have opposite effects on pituitary gonadotropin secretion: inhibin diminishes FSH production but does not affect the release of LH; activin stimulates FSH but not LH.
GnRH, when it reaches the anterior lobe of the pituitary, stimulates the production of LH and FSH from the gonadotrophs in the pituitary gland. GnRH only acts to stimulate the production of the gonadotropic hormones. The periodic release of LH and FSH by the pituitary is responsive not only to GnRH but also to feedback systems involving the target organ of these hormones, the ovary. LH acts primarily on the thecal cells of the ovary to induce the synthesis of steroids, while FSH acts primarily on the granulosa cells of the ovary to stimulate the growth of the ovarian follicles. Both types of ovarian cells are thought to be involved in estrogen production. LH acts on the thecal cells to produce the androgens androstenedione and testosterone, which in turn are transported to the granulosa cells where they are aromatized to form the estrogens estrone and estradiol. Before puberty, FSH release is greater than LH. With the onset of the menstrual cycle, LH secretion is greater than FSH secretion. Increased levels of estradiol and inhibin during the years of menstruation act to inhibit FSH release. After menopause, FSH release again exceeds LH release.
Growth factors produced in the ovary provide means for regulating the hormonal behavior of this gland. Inhibin and activin in particular are related to FSH release. Inhibin is regulated positively by FSH levels. Inhibin preferentially affects FSH release over LH release. Levels of inhibin are observed to decrease dramatically during perimenopause, suggesting that this substance has a permissive role in the elevation of FSH before menopause. Activin is observed to stimulate FSH release. Other growth factors have been identified as having hormonal, autocrine, and paracrine effects within the ovary. Comprehensive Gynecology (eds. Mishell et al. 1997).
Cytokines are involved in the production of reproductive hormones via their activities within the brain and in the ovary. Interleukin-1 (IL-1) is a multifunctional cytokine implicated in a number of aspects of ovarian biology. IL-1 has been implicated in follicular development and atresia, ovulation, steroidogenesis and corpus luteum function. Terranova et al., Am. J. Reprod. Immunol. 37:50-63, 1997. IL-1, when found within the ovary, may be produced by immune and non-immune cells. Machelon et al., Hum. Reprod. 10:2198-03, 1995. IL-1 is involved in rescuing ovarian follicles from apoptosis. Kaipia et al., Annu. Rev. Physiol. 59:349-63, 1997. Conversely, the IL-1 receptor antagonist (IL-1RA) has been shown to block ovulation in vivo and in vitro. Tsafriri, Adv. Exp. Med. Biol. 377:121-40, 1995. Furthermore, IL-1 has been identified as a neurotransmitter, active in releasing norepinephrine, dopamine and serotonin, and affected by their intrahypothalamic levels. Tringali et al., Pharmacol. Res. 36:269-73, 1997. IL-1RA exerts a blocking effect by competitively inhibiting the binding of IL-1 to its receptors. Shintani et al., Mol. Neurobiol. 10:47-71, 1995. Through both central and end-organ mechanisms, cytokines and growth factors have been shown to be implicated in the regulation of reproductive endocrinology.
The age of physiological menopause is understood to have a genetic component. Cramer et al., Fertil. Steril. 64:740-45, 1995; Snieder et al., J. Clin. Endocrinol. and Metab. 83:1875-80, 1998. Because management of menopausal symptoms, prevention of post-menopausal health problems, and diagnosis of early post-menopausal illness form an important part of primary medical care, the ability to determine a genetic predisposition to EOM and to identify causative mutations would be valuable.
In one aspect, the present invention provides a novel method for identifying whether a woman is predisposed to developing early-onset menopause (EOM). In one embodiment, the method comprises determining whether an EOM associated allele is present in a nucleic acid sample obtained from a woman. In a preferred embodiment, the EOM associated allele is IL-1RN (+2018) allele 2 or an allele of the IL-1 (44112332) haplotype.
In certain embodiments, detection of an EOM associated allele may be accomplished directly, e.g. by analyzing the DNA, or indirectly, e.g. by analyzing the RNA or protein products of the DNA. Where the marker in question results in the translation of a mutant protein, the protein can be detected by any of a variety of protein detection methods. Such methods include immunodetection and biochemical tests, such as an activity assay, or size fractionation, where the protein has a change in apparent molecular weight either through truncation, elongation, altered folding or altered post-translational modifications.
In a preferred embodiment, the EOM associated allele can be detected by any of a variety of available techniques, including: 1) performing a hybridization reaction between a nucleic acid sample and a probe that is capable of hybridizing to the allele; 2) sequencing at least a portion of the allele; or 3) determining the electrophoretic mobility of the allele or fragments thereof (e.g., fragments generated by endonuclease digestion). The allele can optionally be subjected to an amplification step prior to performance of the detection step. Preferred amplification methods are selected from the group consisting of: the polymerase chain reaction (PCR), the ligase chain reaction (LCR), strand displacement amplification (SDA), cloning, and variations of the above (e.g. RT-PCR and allele specific amplification). Oligonucleotides necessary for amplification may be selected from anywhere in the IL-1 gene loci, either flanking the marker of interest (as required for PCR amplification) or directly overlapping the marker (as in allele-specific oligonuceotide hybridization). The DNA in the human IL-1 region has been mapped, and oligonucleotides for primers can easily be selected with a commercially available primer selection program. In a particularly preferred embodiment, the sample is hybridized with a set of primers, which hybridize 5xe2x80x2 and 3xe2x80x2 in a sense or antisense sequence to the EOM associated allele, and is subjected to a PCR amplification.
In another aspect, the invention features kits for performing the above-described assays. The kit can include nucleic acid sample collection means and a means for determining whether a subject carries an EOM associated allele. The kit may also comprise control samples, either negative or positive, or standards and/or an algorithmic device for assessing the results, and addition reagents and components, including DNA amplification reagents, DNA polymerases, nucleic acid purification reagents, restriction enzymes, buffers, a nucleic acid sampling device, deoxynucleotides, etc. Information obtained using the assays and kits described herein is useful for example for family planning and for treating or preventing the development of symptoms, which are associated with menopause (e.g. osteoporosis and coronary artery disease). In addition, the information can allow a more customized approach to delaying the onset of or treating the symptoms associated with EOM. For example, this information can enable a doctor to: 1) more effectively prescribe a drug that will address the molecular basis of EOM in the subject; and/or 2) better determine the appropriate dosage of a particular drug for the particular patient.
In another aspect, the invention provides in vitro and in vivo methods for identifying is biomarkers that are useful in monitoring a subject""s progress towards and through menopause. In preferred embodiments, such biomarkers vary depending on a subject""s IL-1 genotype. In certain embodiments, such biomarkers maybe identified by comparing said biomarkers in subjects with an EOM-associated genotype to those in subjects with a genotype not associated with EOM. In certain embodiments, biomarkers may be used to monitor a subject""s progress towards and through menopause.
In another embodiment, the invention features transgenic non-human animals and their uses in identifying biomarkers that are useful in monitoring a subject""s progress towards and through menopause. In yet another embodiment, the transgenic animals may be used to screen for EOM therapeutics. In a preferred variation, such animals may be used to identify agonists and antagonists of IL-1 xcex1 and/or xcex2 activity or agonists and antagonists of IL-1RA activity.
In still another aspect, the invention provides in vitro and in vivo assays for screening test substances to identify EOM therapeutics. In one embodiment, the screening assay comprises contacting a cell or subject comprising an EOM associated IL-1 allele with a test substance. One or more biomarker is observed and changes in one or more biomarker from an EOM-associated phenotype to a non-EOM-associated phenotype indicates that the test substance is likely to be effective as an EOM therapeutic. In preferred embodiments, the one or more biomarker is an IL-1 bioactivity. In yet a further aspect, the invention features methods for treating or preventing the development of early onset menopause in a woman, by administering to the woman, a pharmaceutically effective amount of an EOM therapeutic of the invention.
Other features and advantages of the invention will be apparent from the following detailed description and claims.